mkLTG: a command-line tool for taxonomic assignment of metabarcoding sequences using variable identity thresholds.

Metabarcoding is now a widely used method for biodiversity studies. Taxonomic assignment of environmental sequences is one of the key steps of metabarcoding. Assignments based on lowest common ancestor (LCA) method generally rely on fixed arbitrary thresholds, and this is generally not well adapted for assignment of taxonomically diverse groups with variable coverage in reference databases.

Scientific history, biogeography, and biological traits predict presence of cryptic or overlooked species.

Genetic data show that many nominal species are composed of more than one biological species, and thus contain cryptic species in the broad sense (including overlooked species). When ignored, cryptic species generate confusion which, beyond biodiversity or vulnerability underestimation, blurs our understanding of ecological and evolutionary processes and may impact the soundness of decisions in conservation or medicine. However, very few hypotheses have been tested about factors that predispose a taxon to contain cryptic or overlooked species.

Genetic diversity of the submerged macrophyte depends on habitat hydrology and habitat fragmentation.

The adaptability of plant populations to a changing environment depends on their genetic diversity, which in turn is influenced by the degree of sexual reproduction and gene flow from distant areas. Aquatic macrophytes can reproduce both sexually and asexually, and their reproductive fragments are spread in various ways (e.g.

VTAM: A robust pipeline for validating metabarcoding data using controls.

To obtain accurate estimates for biodiversity and ecological studies, metabarcoding studies should be carefully designed to minimize both false positive (FP) and false negative (FN) occurrences. Internal controls (mock samples and negative controls), replicates, and overlapping markers allow controlling metabarcoding errors but current metabarcoding software packages do not explicitly integrate these additional experimental data to optimize filtering. We have developed the metabarcoding analysis software VTAM, which uses explicitly these elements of the experimental design to find optimal parameter settings that minimize FP and FN occurrences.

COInr and mkCOInr: Building and customizing a nonredundant barcoding reference database from BOLD and NCBI using a semi-automated pipeline.

Reference databases with wide taxonomic coverage are greatly needed in many fields of biology, most particularly for the taxonomic assignment of metabarcoding sequences. Therefore, it is fundamental to be able to access and pool data from different primary databases. The COInr database is a freely available, easy-to-access database of COI reference sequences extracted from the BOLD and NCBI nucleotide databases.

Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies.

Background: In metabarcoding analyses, the taxonomic assignment is crucial to place sequencing data in biological and ecological contexts. This fundamental step depends on a reference database, which should have a good taxonomic coverage to avoid unassigned sequences. However, this goal is rarely achieved in many geographic regions and for several taxonomic groups.

DNA metabarcoding suggests adaptive seasonal variation of individual trophic traits in a critically endangered fish.

Dietary studies are critical for understanding foraging strategies and have important applications in conservation and habitat management. We applied a robust metabarcoding protocol to characterize the diet of the critically endangered freshwater fish Zingel asper (the Rhone streber). We conducted modelling and simulation analyses to identify and characterize some of the drivers of individual trophic trait variation in this species.

Development and characterization of novel SSR markers in the endangered endemic species .

Premise: (Apiaceae) is a polycarpic, perennial herb with a very limited range and small populations. It is listed as "endangered" on the IUCN Red List of Threatened Species. Microsatellite markers can contribute to conservation efforts by allowing the study of the genetic structure of its shrinking populations.

Isolation and characterization of 15 SSR loci for the endangered European tetraploid species (Iridaceae).

Premise: (Iridaceae) is an endangered European perennial tetraploid herb with special conservation interest in the European Union. Microsatellite markers can serve as effective tools for the conservation genetics of this species.

Methods And Results: We utilized a 454 pyrosequencing approach to identify simple sequence repeat (SSR) regions in a microsatellite-enriched library.

One-locus-several-primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies.

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets' complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants).

A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies.

The main objective of this work was to develop and validate a robust and reliable "from-benchtop-to-desktop" metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to faecal DNA samples of an invertebrate-eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage.

QDD version 3.1: a user-friendly computer program for microsatellite selection and primer design revisited: experimental validation of variables determining genotyping success rate.

Microsatellite marker development has been greatly simplified by the use of high-throughput sequencing followed by in silico microsatellite detection and primer design. However, the selection of markers designed by the existing pipelines depends either on arbitrary criteria, or older studies on PCR success. Based on wet laboratory experiments, we have identified the following factors that are most likely to influence genotyping success rate: alignment score between the primers and the amplicon; the distance between primers and microsatellites; the length of the PCR product; target region complexity and the number of reads underlying the sequence.

Microsatellite markers from the Ion Torrent: a multi-species contrast to 454 shotgun sequencing.

The development and screening of microsatellite markers have been accelerated by next-generation sequencing (NGS) technology and in particular GS-FLX pyro-sequencing (454). More recent platforms such as the PGM semiconductor sequencer (Ion Torrent) offer potential benefits such as dramatic reductions in cost, but to date have not been well utilized. Here, we critically compare the advantages and disadvantages of microsatellite development using PGM semiconductor sequencing and GS-FLX pyro-sequencing for two gymnosperm (a conifer and a cycad) and one angiosperm species.

Breakdown of phylogenetic signal: a survey of microsatellite densities in 454 shotgun sequences from 154 non model eukaryote species.

Microsatellites are ubiquitous in Eukaryotic genomes. A more complete understanding of their origin and spread can be gained from a comparison of their distribution within a phylogenetic context. Although information for model species is accumulating rapidly, it is insufficient due to a lack of species depth, thus intragroup variation is necessarily ignored.

A shot in the genome: how accurately do shotgun 454 sequences represent a genome?

Background: Next generation sequencing (NGS) provides a valuable method to quickly obtain sequence information from non-model organisms at a genomic scale. In principle, if sequencing is not targeted for a genomic region or sequence type (e.g.

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries.

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms.

Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing.

Background: The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences.

SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing.

Summary: Characterizing genetic diversity through genotyping short amplicons is central to evolutionary biology. Next-generation sequencing (NGS) technologies changed the scale at which these type of data are acquired. SESAME is a web application package that assists genotyping of multiplexed individuals for several markers based on NGS amplicon sequencing.

Representativeness of microsatellite distributions in genomes, as revealed by 454 GS-FLX titanium pyrosequencing.

Background: Microsatellites are markers of choice in population genetics and genomics, as they provide useful insight into patterns and processes as diverse as genome evolutionary dynamics and demographic processes. The acquisition of microsatellites through multiplex-enriched libraries and 454 GS-FLX Titanium pyrosequencing is a promising new tool for the isolation of new markers in unknown genomes. This approach can also be used to evaluate the extent to which microsatellite-enriched libraries are representative of the genome from which they were isolated.

Cross-species amplification of 41 microsatellites in European cyprinids: A tool for evolutionary, population genetics and hybridization studies.

Background: Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date.

Findings: Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma and Leuciscus leuciscus) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species.

QDD: a user-friendly program to select microsatellite markers and design primers from large sequencing projects.

Summary: QDD is an open access program providing a user-friendly tool for microsatellite detection and primer design from large sets of DNA sequences. The program is designed to deal with all steps of treatment of raw sequences obtained from pyrosequencing of enriched DNA libraries, but it is also applicable to data obtained through other sequencing methods, using FASTA files as input. The following tasks are completed by QDD: tag sorting, adapter/vector removal, elimination of redundant sequences, detection of possible genomic multicopies (duplicated loci or transposable elements), stringent selection of target microsatellites and customizable primer design.

Plasticity of laccase generated by homeologous recombination in yeast.

Laccase-encoding sequences sharing 65-71% identity were shuffledin vivo by homeologous recombination. Yeast efficiently repaired linearized plasmids containing clac1, clac2 or clac5 Trametes sp. C30 cDNAs using a clac3 PCR fragment.

Microsatellite flanking region similarities among different loci within insect species.

Although microsatellites are ubiquitous in eukaryota, the number of available markers varies strongly among taxa. This meta-analysis was conducted on 32 insect species. Sequences were obtained from two assembled whole genomes, whole genome shotgun (WGS) sequences from 10 species and screening partial genomic libraries for microsatellites from 23 species.

Allozyme variation in Parnassius mnemosyne (L.) (Lepidoptera) populations in North-East Hungary: variation within a subspecies group.

Allozyme polymorphism was studied in 11 Parnassius mnemosyne (Linnaeus, 1758) populations in North-East Hungary. Significant departures from Hardy-Weinberg equilibrium were observed in several cases due to heterozygote deficiency. Genetic variability did not display geographical pattern; the level of genetic differentiation was similar between adjacent populations and between populations originating from different geographical regions.

Role of cell wall-degrading enzymes in pathogenicity of Fusarium oxysporum.

Fusarium oxysporum invades its host plants through the roots and colonizes the vascular system. It produces a great variety of cell-wall degrading enzymes (CWDE), such as cellulases, xylanases, pectinases and proteases. Our group has purified and characterized an endopolygalacturonase (PG1), two exopolygalacturonases (PG2 and PG3), an endoxylanase (XYL1) and an endo pectatelyase (PL1).